PROJECT SUMMARY Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, provide a unique combination of infinite self-renewal potential and pluripotency, two properties which impart a powerful system for generating normal human somatic cells for developmental studies, toxicity testing, and cellular therapies. Brain microvascular endothelial cells (BMECs) are a particularly promising cell type that can be derived from hPSCs since BMECs cannot easily be obtained from human tissue or adult stem cells and are of tremendous importance in neurological disease and pharmaceutical evaluation of transport across the blood-brain barrier (BBB). Recently, our team developed a protocol to differentiate hPSCs to BMECs by co- differentiating a mixed population of neural and endothelial progenitors, then selectively subculturing the endothelial progenitors, which acquire BMEC phenotypes. These hPSC-derived BMECs express brain-specific markers including tight junction proteins and molecular transporters. When co-cultured with astrocytes, hPSC- derived BMEC monolayers generate transendothelial electrical resistance comparable to that found in vivo and exhibit polarized transport of nutrients and drugs that correlate with BBB transport in an animal model. These hPSC-derived BMECs provide the first in vitro human BBB model that recapitulates key in vivo BBB phenotypes, and provide a novel platform for understanding BMEC development and regulation. However, the hPSC-derived BMECs lack in vivo levels of BBB marker expression and transporter activity, perhaps as a consequence of the in vitro differentiation microenvironment failing to incorporate key cues present during BBB development. Several studies have implicated fluid flow as an important regulator of vascular function, including barrier formation in BMECs. In this proposal we will test the hypothesis that shear stress provides inductive cues on BBB differentiation at specific developmental stages and is important in maintaining the differentiated phenotypes of hPSC-derived BMECs. Our team's expertise in mechanotransduction, pluripotent stem cell biology, and BBB modeling will permit us to systematically assess the role of shear stress on BMEC differentiation and maintenance of BBB phenotypes. This study will then motivate further mechanistic research in mechanotransduction during BBB development and lead to improvements in human BBB modeling for drug screening applications. Our specific aims to test the hypothesis of this proposal are: 1. Identify stage-specific effects of shear stress on differentiation fates of BMECs and BMEC progenitors 2. Ascertain the effects of shear stress on hPSC-derived BMEC phenotype induction and maintenance 3. Determine the roles of PECAM-1 and VE-cadherin in shear-induced differentiation of BMECs